Abstract：
Are epididymosomes implicated in protein transfer from the epididymis to spermatozoa?
We characterized the contribution of epididymal secretions to the sperm proteome and demonstrated that sperm acquire epididymal proteins through epididymosomes.
Testicular sperm are immature cells unable to fertilize an oocyte. After leaving the testis, sperm transit along the epididymis to acquire motility and fertilizing abilities. It is well known that marked changes in the sperm proteome profile occur during epididymal maturation. Since the sperm is a transcriptional and translational inert cell, previous studies have shown that sperm incorporate proteins, RNA and lipids from extracellular vesicles (EVs), released by epithelial cells lining the male reproductive tract.
We examined the contribution of the epididymis to the post-testicular maturation of spermatozoa, via the production of EVs named epididymosomes, released by epididymal epithelial cells. An integrative analysis using both human and mouse data was performed to identify sperm proteins with a potential epididymis-derived origin. Testes and epididymides from adult humans (n = 9) and adult mice (n = 3) were used to experimentally validate the tissue localization of four selected proteins using high-resolution confocal microscopy. Mouse epididymal sperm were co-incubated with carboxyfluorescein succinimidyl ester (CFSE)-labeled epididymosomes (n = 4 mice), and visualized using high-resolution confocal microscopy.
Adult (12-week-old) C57BL/CBAF1 wild-type male mice and adult humans were used for validation purposes. Testes and epididymides from both mice and humans were obtained and processed for immunofluorescence. Mouse epididymal sperm and mouse epididymosomes were obtained from the epididymal cauda segment. Fluorescent epididymosomes were obtained after labeling the epididymal vesicles with CFSE dye followed by epididymosome isolation using a density cushion. Immunofluorescence was performed following co-incubation of sperm with epididymosomes in vitro. High-resolution confocal microscopy and 3D image reconstruction were used to visualize protein localization and sperm-epididymosomes interactions.
Through in silico analysis, we first identified 25 sperm proteins with a putative epididymal origin that were conserved in both human and mouse spermatozoa. From those, the epididymal origin of four sperm proteins (SLC27A2, EDDM3B, KRT19 and WFDC8) was validated by high-resolution confocal microscopy. SLC27A2, EDDM3B, KRT19 and WFDC8 were all detected in epithelial cells lining the human and mouse epididymis, and absent from human and mouse seminiferous tubules. We found region-specific expression patterns of these proteins throughout the mouse epididymides. In addition, while EDDM3B, KRT19 and WFDC8 were detected in both epididymal principal and clear cells (CCs), SLC27A2 was exclusively expressed in CCs. Finally, we showed that CFSE-fluorescently labeled epididymosomes interact with sperm in vitro and about 12-36% of the epididymosomes contain the targeted sperm proteins with an epididymal origin.
N/A.
The human and mouse sample size was limited and our results were descriptive. The analyses of epididymal sperm and epididymosomes were solely performed in the mouse model due to the difficulties in obtaining epididymal luminal fluid human samples. Alternatively, human ejaculated sperm and seminal EVs could not be used because ejaculated sperm have already contacted with the fluids secreted by the male accessory sex glands, and seminal EVs contain other EVs in addition to epididymosomes, such as the abundant prostate-derived EVs.
Our findings indicate that epididymosomes are capable of providing spermatozoa with a new set of epididymis-derived proteins that could modulate the sperm proteome and, subsequently, participate in the post-testicular maturation of sperm cells. Additionally, our data provide further evidence of the novel role of epididymal CCs in epididymosome production. Identifying mechanisms by which sperm mature to acquire their fertilization potential would, ultimately, lead to a better understanding of male reproductive health and may help to identify potential therapeutic strategies to improve male infertility.
This work was supported by the Spanish Ministry of Economy and Competitiveness (Ministerio de Economía y Competividad; fondos FEDER \'una manera de hacer Europa\' PI13/00699 and PI16/00346 to R.O.; and Sara Borrell Postdoctoral Fellowship, Acción Estratégica en Salud, CD17/00109 to J.C.), by National Institutes of Health (grants HD040793 and HD069623 to S.B., grant HD104672-01 to M.A.B.), by the Spanish Ministry of Education, Culture and Sports (Ministerio de Educación, Cultura y Deporte para la Formación de Profesorado Universitario, FPU15/02306 to F.B.), by a Lalor Foundation Fellowship (to F.B. and M.A.B.), by the Government of Catalonia (Generalitat de Catalunya, pla estratègic de recerca i innovació en salut, PERIS 2016-2020, SLT002/16/00337 to M.J.), by Fundació Universitària Agustí Pedro i Pons (to F.B.), and by the American Society for Biochemistry and Molecular Biology (PROLAB Award from ASBMB/IUBMB/PABMB to F.B.). Confocal microscopy and transmission electron microscopy was performed in the Microscopy Core facility of the Massachusetts General Hospital (MGH) Center for Systems Biology/Program in Membrane Biology which receives support from Boston Area Diabetes and Endocrinology Research Center (BADERC) award DK57521 and Center for the Study of Inflammatory Bowel Disease grant DK43351. The Zeiss LSM800 microscope was acquired using an NIH Shared Instrumentation Grant S10-OD-021577-01. The authors have no conflicts of interest to declare.
We characterized the contribution of epididymal secretions to the sperm proteome and demonstrated that sperm acquire epididymal proteins through epididymosomes.
Testicular sperm are immature cells unable to fertilize an oocyte. After leaving the testis, sperm transit along the epididymis to acquire motility and fertilizing abilities. It is well known that marked changes in the sperm proteome profile occur during epididymal maturation. Since the sperm is a transcriptional and translational inert cell, previous studies have shown that sperm incorporate proteins, RNA and lipids from extracellular vesicles (EVs), released by epithelial cells lining the male reproductive tract.
We examined the contribution of the epididymis to the post-testicular maturation of spermatozoa, via the production of EVs named epididymosomes, released by epididymal epithelial cells. An integrative analysis using both human and mouse data was performed to identify sperm proteins with a potential epididymis-derived origin. Testes and epididymides from adult humans (n = 9) and adult mice (n = 3) were used to experimentally validate the tissue localization of four selected proteins using high-resolution confocal microscopy. Mouse epididymal sperm were co-incubated with carboxyfluorescein succinimidyl ester (CFSE)-labeled epididymosomes (n = 4 mice), and visualized using high-resolution confocal microscopy.
Adult (12-week-old) C57BL/CBAF1 wild-type male mice and adult humans were used for validation purposes. Testes and epididymides from both mice and humans were obtained and processed for immunofluorescence. Mouse epididymal sperm and mouse epididymosomes were obtained from the epididymal cauda segment. Fluorescent epididymosomes were obtained after labeling the epididymal vesicles with CFSE dye followed by epididymosome isolation using a density cushion. Immunofluorescence was performed following co-incubation of sperm with epididymosomes in vitro. High-resolution confocal microscopy and 3D image reconstruction were used to visualize protein localization and sperm-epididymosomes interactions.
Through in silico analysis, we first identified 25 sperm proteins with a putative epididymal origin that were conserved in both human and mouse spermatozoa. From those, the epididymal origin of four sperm proteins (SLC27A2, EDDM3B, KRT19 and WFDC8) was validated by high-resolution confocal microscopy. SLC27A2, EDDM3B, KRT19 and WFDC8 were all detected in epithelial cells lining the human and mouse epididymis, and absent from human and mouse seminiferous tubules. We found region-specific expression patterns of these proteins throughout the mouse epididymides. In addition, while EDDM3B, KRT19 and WFDC8 were detected in both epididymal principal and clear cells (CCs), SLC27A2 was exclusively expressed in CCs. Finally, we showed that CFSE-fluorescently labeled epididymosomes interact with sperm in vitro and about 12-36% of the epididymosomes contain the targeted sperm proteins with an epididymal origin.
N/A.
The human and mouse sample size was limited and our results were descriptive. The analyses of epididymal sperm and epididymosomes were solely performed in the mouse model due to the difficulties in obtaining epididymal luminal fluid human samples. Alternatively, human ejaculated sperm and seminal EVs could not be used because ejaculated sperm have already contacted with the fluids secreted by the male accessory sex glands, and seminal EVs contain other EVs in addition to epididymosomes, such as the abundant prostate-derived EVs.
Our findings indicate that epididymosomes are capable of providing spermatozoa with a new set of epididymis-derived proteins that could modulate the sperm proteome and, subsequently, participate in the post-testicular maturation of sperm cells. Additionally, our data provide further evidence of the novel role of epididymal CCs in epididymosome production. Identifying mechanisms by which sperm mature to acquire their fertilization potential would, ultimately, lead to a better understanding of male reproductive health and may help to identify potential therapeutic strategies to improve male infertility.
This work was supported by the Spanish Ministry of Economy and Competitiveness (Ministerio de Economía y Competividad; fondos FEDER \'una manera de hacer Europa\' PI13/00699 and PI16/00346 to R.O.; and Sara Borrell Postdoctoral Fellowship, Acción Estratégica en Salud, CD17/00109 to J.C.), by National Institutes of Health (grants HD040793 and HD069623 to S.B., grant HD104672-01 to M.A.B.), by the Spanish Ministry of Education, Culture and Sports (Ministerio de Educación, Cultura y Deporte para la Formación de Profesorado Universitario, FPU15/02306 to F.B.), by a Lalor Foundation Fellowship (to F.B. and M.A.B.), by the Government of Catalonia (Generalitat de Catalunya, pla estratègic de recerca i innovació en salut, PERIS 2016-2020, SLT002/16/00337 to M.J.), by Fundació Universitària Agustí Pedro i Pons (to F.B.), and by the American Society for Biochemistry and Molecular Biology (PROLAB Award from ASBMB/IUBMB/PABMB to F.B.). Confocal microscopy and transmission electron microscopy was performed in the Microscopy Core facility of the Massachusetts General Hospital (MGH) Center for Systems Biology/Program in Membrane Biology which receives support from Boston Area Diabetes and Endocrinology Research Center (BADERC) award DK57521 and Center for the Study of Inflammatory Bowel Disease grant DK43351. The Zeiss LSM800 microscope was acquired using an NIH Shared Instrumentation Grant S10-OD-021577-01. The authors have no conflicts of interest to declare.
摘要：
附睾体是否与从附睾到精子的蛋白质转移有关？
我们表征了附睾分泌物对精子蛋白质组的贡献，并证明了精子通过附睾体获得附睾蛋白质。
睾丸精子是不能使卵母细胞受精的未成熟细胞。离开睾丸后，精子沿着附睾运输以获得运动性和受精能力。众所周知,精子蛋白质组谱在附睾成熟期间发生显著变化。由于精子是转录和翻译惰性细胞，以前的研究表明，精子含有蛋白质，来自细胞外囊泡（EV）的RNA和脂质，由雄性生殖道内衬的上皮细胞释放。
我们检查了附睾对精子睾丸后成熟的贡献，通过生产名为附睾小体的电动汽车，由附睾上皮细胞释放。使用人和小鼠数据进行综合分析以鉴定具有潜在附睾来源的精子蛋白。使用高分辨率共聚焦显微镜，使用成年人（n=9）和成年小鼠（n=3）的睾丸和附睾实验验证了四种选定蛋白质的组织定位。将小鼠附睾精子与羧基荧光素琥珀酰亚胺酯（CFSE）标记的附睾体（n=4小鼠）共孵育，并使用高分辨率共聚焦显微镜进行可视化。
成年(12周龄)C57BL/CBAF1野生型雄性小鼠和成人用于验证目的。获得小鼠和人类的睾丸和附睾并进行免疫荧光处理。从附睾尾段获得小鼠附睾精子和小鼠附睾小体。用CFSE染料标记附睾囊泡，然后使用密度垫分离附睾囊泡后，获得荧光附睾囊泡。在体外将精子与附睾小体共孵育后进行免疫荧光。高分辨率共聚焦显微镜和3D图像重建用于可视化蛋白质定位和精子-附睾体相互作用。
通过硅片分析，我们首先鉴定出25种精子蛋白，这些蛋白具有推定的附睾起源，在人和小鼠精子中都是保守的。从那些,四种精子蛋白的附睾起源(SLC27A2，EDDM3B，KRT19和WFDC8）通过高分辨率共聚焦显微镜进行验证。SLC27A2,EDDM3B,KRT19和WFDC8均在人和小鼠附睾的上皮细胞中检测到，并且不存在于人和小鼠的生精小管中。我们在整个小鼠附睾中发现了这些蛋白质的区域特异性表达模式。此外,而EDDM3B,在附睾主细胞和透明细胞（CC）中均检测到KRT19和WFDC8，SLC27A2仅在CC中表达。最后,我们发现CFSE荧光标记的附睾体在体外与精子相互作用，约12-36％的附睾体含有具有附睾起源的靶向精子蛋白。
不适用。
人和小鼠样本大小有限，我们的结果是描述性的。由于难以获得附睾腔液人样品，因此仅在小鼠模型中进行附睾精子和附睾体的分析。或者，人类射精的精子和精液不能使用，因为射精的精子已经与男性附属性腺分泌的液体接触，精浆电动汽车除附睾小体外还含有其他电动汽车，例如丰富的前列腺衍生的电动汽车。
我们的发现表明附睾体能够为精子提供一组新的附睾衍生的蛋白质，这些蛋白质可以调节精子蛋白质组，随后,参与精子细胞的睾丸后成熟。此外,我们的数据为附睾CCs在附睾小体产生中的新作用提供了进一步的证据.确定精子成熟以获得受精潜力的机制，最终,有助于更好地了解男性生殖健康，并可能有助于确定改善男性不育的潜在治疗策略。
这项工作得到了西班牙经济和竞争力部的支持（MinisteriodeEconmíayCompetitivedad；fondosFEDER\'unamaneradehacerEuropa\'PI13/00699和PI16/00346给R.O.；和SaraBorrell博士后奖学金，EstratégicaenSalud,CD17/00109toJ.C.),由美国国立卫生研究院（授予S.B.HD040793和HD069623，将HD104672-01授予M.A.B.)，西班牙教育部，文化和体育(MinisteriodeEducación,CulturayDeporteparalaFormacióndeProfesoradoUniversitario,FPU15/02306至F.B.），由Lalor基金会奖学金（F.B.和M.A.B.），由加泰罗尼亚政府（GeneralitatdeCatalunya，解放军埃斯特雷克·德·科·科·安·萨特，PERIS2016-2020，SLT002/16/00337至M.J.)，作者：FundacióUniversityAgustíPedroiPons（至F.B.），和美国生物化学和分子生物学学会（PROLAB奖从ASBMB/IUBMB/PABMB到F.B.）。在马萨诸塞州总医院（MGH）系统生物学/膜生物学计划中心的显微镜核心设施中进行了共聚焦显微镜和透射电子显微镜检查，该中心获得了波士顿地区糖尿病和内分泌学研究中心（BADERC）奖DK57521和炎症性肠病研究中心授予DK43351的支持。蔡司LSM800显微镜是使用NIH共享仪器授权S10-OD-021577-01获得的。作者没有利益冲突要声明。
我们表征了附睾分泌物对精子蛋白质组的贡献，并证明了精子通过附睾体获得附睾蛋白质。
睾丸精子是不能使卵母细胞受精的未成熟细胞。离开睾丸后，精子沿着附睾运输以获得运动性和受精能力。众所周知,精子蛋白质组谱在附睾成熟期间发生显著变化。由于精子是转录和翻译惰性细胞，以前的研究表明，精子含有蛋白质，来自细胞外囊泡（EV）的RNA和脂质，由雄性生殖道内衬的上皮细胞释放。
我们检查了附睾对精子睾丸后成熟的贡献，通过生产名为附睾小体的电动汽车，由附睾上皮细胞释放。使用人和小鼠数据进行综合分析以鉴定具有潜在附睾来源的精子蛋白。使用高分辨率共聚焦显微镜，使用成年人（n=9）和成年小鼠（n=3）的睾丸和附睾实验验证了四种选定蛋白质的组织定位。将小鼠附睾精子与羧基荧光素琥珀酰亚胺酯（CFSE）标记的附睾体（n=4小鼠）共孵育，并使用高分辨率共聚焦显微镜进行可视化。
成年(12周龄)C57BL/CBAF1野生型雄性小鼠和成人用于验证目的。获得小鼠和人类的睾丸和附睾并进行免疫荧光处理。从附睾尾段获得小鼠附睾精子和小鼠附睾小体。用CFSE染料标记附睾囊泡，然后使用密度垫分离附睾囊泡后，获得荧光附睾囊泡。在体外将精子与附睾小体共孵育后进行免疫荧光。高分辨率共聚焦显微镜和3D图像重建用于可视化蛋白质定位和精子-附睾体相互作用。
通过硅片分析，我们首先鉴定出25种精子蛋白，这些蛋白具有推定的附睾起源，在人和小鼠精子中都是保守的。从那些,四种精子蛋白的附睾起源(SLC27A2，EDDM3B，KRT19和WFDC8）通过高分辨率共聚焦显微镜进行验证。SLC27A2,EDDM3B,KRT19和WFDC8均在人和小鼠附睾的上皮细胞中检测到，并且不存在于人和小鼠的生精小管中。我们在整个小鼠附睾中发现了这些蛋白质的区域特异性表达模式。此外,而EDDM3B,在附睾主细胞和透明细胞（CC）中均检测到KRT19和WFDC8，SLC27A2仅在CC中表达。最后,我们发现CFSE荧光标记的附睾体在体外与精子相互作用，约12-36％的附睾体含有具有附睾起源的靶向精子蛋白。
不适用。
人和小鼠样本大小有限，我们的结果是描述性的。由于难以获得附睾腔液人样品，因此仅在小鼠模型中进行附睾精子和附睾体的分析。或者，人类射精的精子和精液不能使用，因为射精的精子已经与男性附属性腺分泌的液体接触，精浆电动汽车除附睾小体外还含有其他电动汽车，例如丰富的前列腺衍生的电动汽车。
我们的发现表明附睾体能够为精子提供一组新的附睾衍生的蛋白质，这些蛋白质可以调节精子蛋白质组，随后,参与精子细胞的睾丸后成熟。此外,我们的数据为附睾CCs在附睾小体产生中的新作用提供了进一步的证据.确定精子成熟以获得受精潜力的机制，最终,有助于更好地了解男性生殖健康，并可能有助于确定改善男性不育的潜在治疗策略。
这项工作得到了西班牙经济和竞争力部的支持（MinisteriodeEconmíayCompetitivedad；fondosFEDER\'unamaneradehacerEuropa\'PI13/00699和PI16/00346给R.O.；和SaraBorrell博士后奖学金，EstratégicaenSalud,CD17/00109toJ.C.),由美国国立卫生研究院（授予S.B.HD040793和HD069623，将HD104672-01授予M.A.B.)，西班牙教育部，文化和体育(MinisteriodeEducación,CulturayDeporteparalaFormacióndeProfesoradoUniversitario,FPU15/02306至F.B.），由Lalor基金会奖学金（F.B.和M.A.B.），由加泰罗尼亚政府（GeneralitatdeCatalunya，解放军埃斯特雷克·德·科·科·安·萨特，PERIS2016-2020，SLT002/16/00337至M.J.)，作者：FundacióUniversityAgustíPedroiPons（至F.B.），和美国生物化学和分子生物学学会（PROLAB奖从ASBMB/IUBMB/PABMB到F.B.）。在马萨诸塞州总医院（MGH）系统生物学/膜生物学计划中心的显微镜核心设施中进行了共聚焦显微镜和透射电子显微镜检查，该中心获得了波士顿地区糖尿病和内分泌学研究中心（BADERC）奖DK57521和炎症性肠病研究中心授予DK43351的支持。蔡司LSM800显微镜是使用NIH共享仪器授权S10-OD-021577-01获得的。作者没有利益冲突要声明。
